ERYTHROPOIETIN--BIOCHEMISTRY AND GENE CLONING

Project: Research project

Description

Erythropoietin (Ep) is the glycoprotein hormone which is responsible for
maintaining erythropoiesis in animals and man. Of many putative growth
factors affecting hematopoiesis, Ep is the only one with a defined
physiological role. However, understanding of the mechanisms of Ep's
action on responsive cells has been limited due to 1) insufficient
quantities of pure Ep, 2) lack of appropriate laboratory probes and 3)
difficulties in obtaining sufficiently enriched populations of target cells
for study. We have purified Ep in microgram amounts and we propose to
utilize recent developments in molecular and physiological interactions to
study the biochemistry of Ep and its molecular and cellular-biology with
responsive cells. We will use monoclonal and polyvalent antibodies to Ep
and to defined polypeptide sequences to further purify Ep by immunoaffinity
chromatography. As larger quantities of Ep become available, we will
determine its biochemical properties and amino acid sequence. We will
develop site-specific antibodies to Ep, both polyvalent and monoclonal, to
immunologically characterize the molecule. Functional properties will be
correlated with antibody-defined structural domains. We will clone the Ep
coding sequences from anemic baboon kidney. The cDNA libraries will be
screened by immunological methods using anti-Ep antibodies and
oligonucleotide probes developed from amino acid sequence data. As the
protein sequence is determined, it will be compared to the nucleotide
sequences for Ep coding regions. Concurrently, we will attempt to develop
labeled, biologically active Ep probes using different iodination schemes,
tritium labeling of carbohydrate and amino groups, photoaffinity labeling,
and chemical derivatization. Alternatively, we will use labeled or
flouresceinated antibody or Fab fragments to identify Ep. With the acquisition of additional purified Ep as well as biologically
active probes we will purify populations of Ep-responsive cells and begin
studies to identify the Ep receptor and its properties. From the sequence
of experiments outlined in this proposal we can expect greater
understanding of Ep, its protein structure, its genomic coding sequences,
identification of target cells bearing presumed receptors and
characterization of Ep interactions with the receptor. These studies may
serve as a model for understanding the function of other hematopoietic
growth factors and, ultimately, may lead to therapeutic benefit in
disorders accompanied by Ep deficiency.
StatusFinished
Effective start/end date4/1/853/31/88

Funding

  • National Institutes of Health

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Erythropoietin
Biochemistry
Organism Cloning
Genes
Erythropoietin Receptors
Amino Acid Sequence
Immunoglobulin Fragments
Immunoglobulin Fab Fragments
Tritium
Antibodies
Papio
Health Services Needs and Demand
Erythropoiesis
Halogenation
Hematopoiesis
Gene Library
Cell Biology

ASJC

  • Medicine(all)